A comparative study of the metal ion uptake and antioxidant enzyme activities of Fusarium equiseti and Fusarium acuminatum as a function of external magnesium concentration

The increase of Mg2+, from 1.3 to 3 microM, in growth medium of F. equiseti and F. acuminatum increased intracellular magnesium levels from 0.83 and 0.81 microM to 1.75 and 1.42 microM on the 12th day, respectively. Intracellular magnesium levels also elevated depending upon the number of incubation days. The maximum manganese levels of F. equiseti and F. acuminatum obtained in 1.6 microM Mg2+ culture medium were 0.67 and 1.23 microM, while maximum iron levels were determined to be 1.3 microM Mg2+ as 0.51 and 0.29 microM, respectively. The maximum intracellular iron and manganese levels were decreased significantly with increasing Mg2+ concentration in the culture medium and were increased depending upon the incubation period. However, intracellular zinc levels of these strains didn't change with Mg2+ concentration and incubation period.The maximum superoxide dismutase (MnSOD) activities of F. equiseti and F. acuminatum, related to increased intracellular manganese levels up to 1.6 microM Mg2+ in growth medium, were determined to be 78 and 110 IU/mg, respectively. CAT activity variations showed agreement with SOD activity and reached a maximum at 320 and 225 IU/mg under the same conditions. The minimum LPO levels of the Fusarium strains with the maximum MnSOD and CAT activities were determined as 1.2 and 0.9 nmol MDA/g., wet weight. The higher LPO level of F. equiseti grown at the same condition, in spite of 1.42-fold higher CAT activity due to the 1.41-fold lower SOD activity, as well as a 2.0-fold higher iron level, indicated increases in the generation of reactive oxygen species via the Fenton reaction.     

Production,purification, characterization,antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL⁻¹ at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg⁻¹ of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL⁻¹) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL⁻¹, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.     

Influence of indole acetic acid on antioxidant levels and enzyme activities of glucose metabolism in rat liver

Indole acetic acid (IAA) is an auxin and can be synthesized in animals. This compound is metabolized in vitro by peroxidase, producing reactive oxygen species. The toxic effect of indole acetic acid in leukocytes is associated with peroxidase activities and these processes have been implicated in activation of glucose and glutamine metabolism. However, studies in vitro have shown that IAA, in absence of peroxidase, is an antioxidant almost as high in potency as those of other indolic compounds. The purpose of this study was to investigate the possible involvement of a toxic effect of indole acetic acid in the liver, as evidenced by oxidative stress and enzyme activities of the glucose pathway. The animals received IAA by subcutaneous or gavage administration in a phosphate buffered saline (the control group received only the phosphate buffered saline). The other groups received IAA at concentrations of 1 mg, 18 mg and 40 mg per kg of body mass per day. Treatments with 18 mg and 40 mg IAA decreased the activity of catalase by both subcutaneous (30% and 26%) or gavage administration (19% and 28%), respectively. A similar effect was observed on the activity of glutathione peroxidase of animals exposed to 18 mg and 40 mg IAA: A decrease of 34% and 29%, respectively, for subcutaneous administration and a decrease of 29% and 25%, respectively, for gavage administration. However, in neither source of administration did the acid alter superoxide dismutase, glutathione reductase and myeloperoxidase activities. Another alteration was observed in respect of reduced glutathione content in this organ. The lipid peroxidation level showed a significant decrease with subcutaneous (30%, 29% and 24%) and gavage administration (25%, 26% and 24%) using 1 mg, 18 mg and 40 mg of IAA, respectively compared with the control. The reduced glutathione content and catalase activity in the plasma were not altered by either of the two methods of administration. In addition to these findings, after subcutaneous or gavage administration of IAA, the activities of hepatic enzymes of glucose metabolism were not affected (glucokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and citrate synthase). Evidence is presented herein that IAA did not have a pro-oxidant effect in the liver as deduced from a reduction of catalase and glutathione peroxidase activities, a decrease of lipid peroxidation content and no alteration of the pool of reduced glutathione. The effects of IAA were independent of the way of administration.     

The effect of NaCl on antioxidant enzyme activities in potato seedlings

The effect of NaCl on the growth and activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were investigated in the seedlings of four potato cultivars (Agria, Kennebec; relatively salt tolerant, Diamant and Ajax; relatively salt sensitive). The shoot fresh mass of Agria and Kennebec did not changed at 50 mM NaCl, whereas in Diamant and Ajax it decreased to 50 % of that in the controls. In Agria and Kennebec, SOD activity increased at 50 mM NaCl, but no significant changes observed in Diamant and Ajax. At higher NaCl concentration, SOD activity reduced in all cultivars. CAT and POD activities increased in all cultivars under salt stress. Unlike the other cultivars, in Ajax seedlings, APX activity increased in response to NaCl stress. We also observed new POD and SOD isoenzyme activities and changes in isoenzyme compositions under salt stress. These results suggest that salt-tolerant potato cultivars may have a better protection against reactive oxygen species (ROS) by increasing the activity of antioxidant enzymes (especially SOD) under salt stress.     

The effects of some antioxidant vitamin- and trace element-supplemented diets on activities of SOD, CAT, GSH-Px and LPO levels in chicken tissues

The effect of diets containing antioxidant vitamins and trace elements on chicken tissue activities of SOD, CAT, GSH-Px and of LPO levels was investigated. Chickens, 45 weeks of age were divided into six groups: control group, Cu group (13.2 mg Cu kg(-1) diet); Se group (0.07 mg Se kg(-l) diet); vitamin E group (70 mg DL-alpha-tocopherol acetate kg(-1) diet) and a constant level vitamin C, 200 mg kg(-1) diet); vitamin A group (240 mg retinol acetate kg(-1) diet) and vitamin C group (500 mg ascorbic acid kg(-1) diet). Significant variation of these antioxidant enzyme activities and LPO levels according to gender was demonstrated statistically. In the Cu group, CuZnSOD activity in the liver, erythrocyte, kidney and heart significantly increased by 75, 40, 12, 12% respectively (P<0.05). MnSOD activity in the heart, liver, kidney and brain of the vitamin C and in the heart of Cu group were found to be increased by approximately 15%, while in liver tissue of the Cu group it was reduced by 19% (P<0.05). GSH-Px activities in the Se, vitamin E and C groups were significantly increased, conversely LPO levels decreased (P<0.001). CAT activities in the liver and heart of the vitamin C group were significantly decreased (by 32%), but in kidney tissue only that of the Cu group was increased from 30.2 +/- 4.767 to 144.49 +/- 6.93 U mg(-1) P<0.001. The resistance to stress of the vitamin E and C groups, which had significantly increased activities of antioxidant enzymes and decreased lipid peroxide levels, were determined in 60% moisture medium at 45 degrees C.     

Nitric oxide levels and antioxidant enzyme activities in jaundices of premature infants

Free radicals are effective in the genesis of several diseases in the neonatal period. This study aimed to show the relationship between serum bilirubin levels and plasma nitric oxide and the activity of enzymes in the erythrocyte such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in premature infants. In the study, 20 premature infants with newborn jaundice were included and the control group was formed by 15 premature infants without jaundice. Venous blood samples were taken from all neonates in the study and control groups on the first day of hospitalization. Plasma nitric oxide levels and activities of SOD, GSH-Px and CAT enzymes in the erythrocytes were investigated in these samples. Plasma nitric oxide and serum bilirubin levels were found to be significantly higher in the study group (47.4 +/- 7.25 micromol l(-1), 18.41 +/- 3.28 mg dl(-1), respectively) than those in the control group (33.46 +/- 6.43 micromol l(-1), 4.35 +/- 0.60 mg dl(-1), respectively; p < 0.001). In addition, erythrocyte SOD, GSH-Px and CAT enzyme activities (724 +/- 78.61, 673 +/- 90.5, 63 +/- 12.8 U g(-1) Hb, respectively) were found to be significantly lower in the study group than those in the control group (1208 +/- 129.04, 1097.6 +/- 75.8, 99.06 +/- 12.4 U g(-1) Hb, respectively, p < 0.001). It was concluded that in the aetiology of hyperbilirubinemia, neonatal erythrocytes and nitric oxide reactions are affected differently and that erythrocyte haemolysis caused as a result of these effects may play a role in the aetiopathogenesis of unconjugated hyperbilirubinemia. Haemolysis may also be seen because of the inadequacy of the protection by erythrocytes against the cytotoxic effects of free radicals resulting from the lack of antioxidant enzymes in these cells.     

The effect of glucose ingestion on basal and induced enzyme levels in rat tissues

The ingestion of glucose ad libitum (for 19 h to 3 days) decreased the levels of enzymes concerned with amino acid metabolism in liver and intestine (but not in kidney and brain) and raised those of hepatic glucokinase, pyruvate kinase and NADP-malate dehydrogenase.Glucose feeding inhibited the substrate and cortisol induction of tryptophan oxygenase; it did not diminish the induction of tyrosine aminotransferase by glucagon (in adrenalectomized rats) or of renal ornithine aminotransferase by estrogen and it enhanced the response of NADP-malate dehydrogenase to thyroxine. In hepatomas, as opposed to normal liver, 24 h of glucose feeding increased the basal and the cortisol-induced levels of tyrosine aminotransferase.The results obtained with endocrinectomized and hormone-treated rats led to the following conclusions: (1) the effects of glucose ingestion on the quantitative pattern of hepatic enzymes is not mediated through altered secretion of pituitary, adrenal or pancreatic hormones but presumably by metabolites of glucose; (2) glucose, ingested in large amounts over 1–3 days, is not an inhibitor of enzyme inductions in general: its effect varies with the enzyme, the inducer and the tissue in which the enzyme is located.     

Variations of alcohol dehydrogenase activity and fermentative pyruvate,ethanol production of F. equiseti and F. acuminatum depend on the yeast extract and urea concentrations

In this study, pyruvate production of Fusarium equiseti was significantly increased when the yeast extract concentration was raised from 5 to 25 g/L while it was increased to only up to 10 g/L yeast extract in F. acuminatum. Upon supplementation with urea as an alternative nitrogen source, production of pyruvate for both of the Fusarium species were decreased with respect to increase in urea concentration in medium. On the other hand, ethanol production and alcohol dehydrogenase activity of F. equiseti were decreased approximately 1.9- and 1.6-fold with an increase in yeast concentration from 5 to 25 whereas the levels of F. acuminatum were increased 2.3- and 1.8-fold, respectively. In addition, ethanol productions and ADH activities in F. equiseti and F. acuminatum significantly increased on the 12th day up to 15 and 25 g/L urea concentrations, respectively. However, they were significantly decreased under these conditions at higher nitrogen sources. In addition, ethanol production and alcohol dehydrogenase activity in urea supplemented medium were higher than yeast extract supplemented. The results may suggest that the pyruvate, ethanol production and ADH enzyme activity variations and balance between aerobic and anaerobic respiration in F. equiseti and F. acuminatum were effected from yeast extract and urea concentrations in the nutrient medium.     

The effect of ovarian hormones on antioxidant enzyme activities in the brain of male rats

The brain is widely responsive to gonadal hormones. The functional significance of ovarian hormones in the brain is evident from biochemical studies indicating that estradiol or progesterone treatment of testectomized rats produces changes of antioxidant enzyme activities. The effect of estradiol benzoate (EB) and progesterone (P) in the control of antioxidant (AO) enzyme activities was studied in the brain of adult male Wistar rats. The activities of catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and glutathione reductase (GR) were measured in appropriate subcellular fractions, prepared from brains of animals belonging to various experimental groups. These groups were designed with the intention to follow changes in enzyme activities 2 h or 24 h after systemic administration of 5 microg EB or 2 mg P to testectomized (TX) animals. The obtained results show that both EB and P increase CAT activity, whereas EB decreases GSH-Px, GST and GR activities. These findings clearly show the modulatory role of EB and P in the control of enzymes responsible for the protection of rat nerve cells against oxidative damage caused by free oxygen radicals.     

The effect of A23187 on glucose production from methylglyoxal and pyruvate in isolated murine hepatocytes.

1. A23187 increased the glucose production from methylglyoxal in isolated hepatocytes, and maximal stimulation was obtained at 10(-6) M. The effect of A23187 was dependent on the presence of Ca2+. 2. Glucose production from pyruvate (less than 1 mM) in isolated hepatocytes was stimulated by A23187 in the presence of 2.5 mM Ca2+ and was depressed at pyruvate concentrations above 1 mM. Both the virtual Km and the virtual Vmax of glucose production from pyruvate were decreased by A23187.     

Effects of soy protein and genistein on blood glucose, antioxidant enzyme activities, and lipid profile in streptozotocin-induced diabetic rats

In the current study, the effect of soy protein and genistein, one of the main isoflavones in soybeans, on blood glucose, lipid profile, and antioxidant enzyme activities in streptozotocin (STZ)-induced diabetic rats was investigated. Male Sprague-Dawley rats were divided into nondiabetic control, STZ, STZ-genistein supplemented group (STZ-G; 600 mg/kg diet), and STZ-isolated soy protein supplemented group (STZ-ISP; 200 g/kg diet). Diabetes was induced by a single injection of STZ (50 mg/kg BW) freshly dissolved in 0.1 mol/L citrate buffer (pH 4.5) into the intraperitonium. Diabetes was confirmed by measuring the fasting blood glucose concentration 48-h post-injection. The rats with blood glucose level above 350 mg/dL were considered to be diabetic. Genistein and ISP were supplemented in the diet for 3 weeks. The supplementation of genistein and ISP increased the plasma insulin level but decreased the HbA(IC) level of the STZ-induced diabetic rats. The supplementation of genistein and ISP increased the glucokinase level of the STZ-induced diabetic rats. A significant reduction in glucose-6-phosphatase was observed in the groups treated with genistein and ISP in comparison with the diabetic control group. Hepatic superoxide dismutase, catalase, and glutathione peroxidase activities of the STZ-induced diabetic rats were significantly decreased in comparison with the control rats. Administering genistein and ISP to the STZ-induced diabetic rats significantly increased those enzyme activities. The concentration of thiobarbituric acid reactive substances in the STZ-induced diabetic rats was significantly elevated, while the genistein and ISP supplement decreased it to the control concentration. Genistein and ISP supplements seem to be beneficial for correcting the hyperglycemia and preventing diabetic complications.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Effect of temperature on secondary metabolites production and antioxidant enzyme activities in Eleutherococcus senticosus somatic embryos

Somatic embryos of Eleutherococcus senticosus were exposed at 12, 16, 24 and 30 °C for duration of 45 days in bioreactor. The effects of such treatments on the growth, eleutheroside B, E, E₁, total phenolics, flavonoids, chlorogenic acid concentrations and antioxidant enzymes activities were investigated. The results revealed that low (12 and 18 °C) and high (30 °C) temperature caused significant decrease in fresh weight (FW), dry weight (DW), total phenolics, flavonoids and total eleutheroside accumulation, while low temperature increased eleutheroside E accumulation in somatic embryos. Low temperature significantly increased superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities whereas a strong increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activity was obtained at 12 °C grown somatic embryos. In contrast, high temperature significantly decreased antioxidant enzymes activities and even guaiacol peroxidase (G-POD) activity also decreased at low temperature in comparison to 24 °C grown embryos. These data suggest that low and high temperature treatment provoked an oxidative stress in E. senticosus embryos, as shown by the increase in lipid peroxidation. The increase in lipid peroxidation was paralleled by a rise in lipoxygenase (LOX) activity and hydrogen peroxide (H₂O₂) content. However, this stress was more prominent at high temperature than low temperature grown embryos. This result suggests that the reduced growth of embryo at 30 °C was concomitant with reduced efficiency of these protective enzymes. On the other hand, increases in antioxidant activities at 12 and 18 °C could also be a response to the cellular damage; however, this increase could not stop the deleterious effects of low temperature, but reduced stress severity thus allowing embryo growth to occur.     

厚壳贻贝抗氧化酶活性和脂质过氧化物含量对Cd和Aroclor 1254胁迫的响应

以厚壳贻贝(Mytilus coruscus)为目标生物,研究了不同浓度Cd2+(0.194、0.388、0.775 mg·L-1)、Aroclor 1254(0.005、0.01、0.05 mg·L-1)单一和复合胁迫对其消化盲囊抗氧化酶活性及脂质过氧化产物丙二醛(MDA)含量的影响。结果表明:(1)单一和复合胁迫均可导致厚壳贻贝抗氧化酶的活性先升高后抑制。高浓度组(0.775 mg·L-1Cd2+、0.05mg·L-1Aroclor 1254)在单一胁迫第1天时,抗氧化酶的活性即达峰值。抗氧化酶活性的下降幅度和速率与胁迫物质作用时间和浓度呈正相关。(2)SOD、GSH-Px对胁迫的敏感性高于CAT。(3)各胁迫处理组MDA含量随胁迫时间延长均显著升高(P0.01)。单一胁迫的效应与剂量和时间呈正相关。(4)复合胁迫的效应强于单一胁迫。推测高于环境水平的Cd2+、Aroclor 1254对厚壳贻贝可产生明显氧化胁迫,抗氧化防御系统可以作为海洋环境重金属-有机物复合污染监测的潜在生物标志物。     

UV-B induced mortality and antioxidant enzyme activities in Daphnia magna at different oxygen concentrations and temperatures

Survival of adult Daphnia magna was assessed after acute (<96h) exposure to UV₃₁₂ under various temperatures (6, 12 and 18°C)or oxygen concentrations (5.6, 8.5 and 14.1 mg O₂ l^–1)in the laboratory. The surviving animals were screened for theenzymes catalase (CAT) and glutathione transferase (GST), whichmay protect against UV-induced oxidative damage. In addition,the same two enzymes were assayed in separate experiments afteracute exposure to UV₃₁₂ (6 h, 0.014 mW cm^–2) and the differentlevels of temperature and oxygen. No differences were observedin either CAT or GST activity after exposure to the three levelsof oxygen, but there was a tendency for decreasing specificactivity with decreasing temperature for both enzymes. CAT activitywas not influenced by UV radiation, whereas GST activity displayeda slight increase. Oxygen concentration did not influence survivalduring UV exposure but, contrary to expectations, survival testsat different temperatures clearly showed that reduced temperatureincreased survival. The results indicate that temperature effectsmust be considered when comparing dose effect relationshipsin situ, and suggest that low temperature is not a major causeof UV susceptibility in cold-adapted alpine and Arctic populationsof Daphnia.     

Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple

The changes of ascorbic acid, dehydroascorbic acid, and glutathione content and related enzyme activities were studied in apple buds during dormancy and thidiazuron-induced bud break. An increase in ascorbic acid, reduced form of glutathione (GSH), total glutathione, total non-protein thiol (NPSH) and non-glutathione thiol (RSH) occurred as a result of induction by thidiazuron during bud break, whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased during the same period. Thidiazuron also enhanced the ratio of GSH/GSSG, and activities of ascorbate free radical reductase (AFR; EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11). dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2). The ascorbic acid content and the activities of AFR, ascorbate peroxidase, and DHAR peaked when buds were in the side green or green tip stage just prior to the start of rapid expansion, and declined thereafter. The GSH, NPSH, RSH, ratio of GSH/GSSG, and activities of GR increased steadily during bud development.